Enhancing the Efficacy of HIPEC Through Bromelain: A Preclinical Investigation in Appendiceal Cancer.

INTRODUCTION: Appendiceal cancer (AC) excessive mucin production is a barrier to heated intraperitoneal chemotherapy (HIPEC) drug delivery. Bromelain is a pineapple stem extract with mucolytic properties. We explored bromelain treatment effects against mucinous AC in a patient-derived tumor organoid (PTO) model and an AC cell line. PATIENTS AND METHODS: PTOs were fabricated from tumor specimens obtained from patients with AC undergoing cytoreductive surgery with HIPEC. PTOs underwent HIPEC treatment with bromelain, cisplatin, and mitomycin C (MMC) at 37 °C and 42 °C with and without bromelain pretreatment. RESULTS: From October 2020 to May 2023, 16 specimens were collected from 13 patients with low-grade (12/16, 75%) and high-grade AC (4/16, 25%). The mucin-depleting effects of bromelain were most significant in combination with N-acetylcysteine (NAC) compared with bromelain (47% versus 10%, p = 0.0009) or NAC alone (47% versus 12.8%, p = 0.0027). Bromelain demonstrated > 31% organoid viability reduction at 60 min (p < 0.001) and > 66% in 48 h (p < 0.0001). Pretreatment with bromelain increased cytotoxicity of both cisplatin and MMC HIPEC conditions by 31.6% (p = 0.0001) and 35.5% (p = 0.0001), respectively. Ki67, CK20, and MUC2 expression decreased after bromelain treatment; while increased caspase 3/7 activity and decreased Bcl-2 (p = 0.009) and Bcl-xL (p = 0.01) suggest induction of apoptosis pathways. Furthermore, autophagy proteins LC3A/B I (p < 0.03) and II (p < 0.031) were increased; while ATG7 (p < 0.01), ATG 12 (p < 0.04), and Becline 1(p < 0.03), expression decreased in bromelain-treated PTOs. CONCLUSIONS: Bromelain demonstrates cytotoxicity and mucolytic activity against appendiceal cancer organoids. As a pretreatment agent, it potentiates the cytotoxicity of multiple HIPEC regimens, potentially mediated through programmed cell death and autophagy.

3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening.

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated "chips." These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber "chips" containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

In Vitro Three-Dimensional (3D) Models for Melanoma Immunotherapy.

Melanoma is responsible for the majority of skin cancer-related fatalities. Immune checkpoint inhibitor (ICI) treatments have revolutionized the management of the disease by significantly increasing patient survival rates. However, a considerable number of tumors treated with these drugs fail to respond or may develop resistance over time. Tumor growth and its response to therapies are critically influenced by the tumor microenvironment (TME); it directly supports cancer cell growth and influences the behavior of surrounding immune cells, which can become tumor-permissive, thereby rendering immunotherapies ineffective. Ex vivo modeling of melanomas and their response to treatment could significantly advance our understanding and predictions of therapy outcomes. Efforts have been directed toward developing reliable models that accurately mimic melanoma in its appropriate tissue environment, including tumor organoids, bioprinted tissue constructs, and microfluidic devices. However, incorporating and modeling the melanoma TME and immune component remains a significant challenge. Here, we review recent literature regarding the generation of in vitro 3D models of normal skin and melanoma and the approaches used to incorporate the immune compartment in such models. We discuss how these constructs could be combined and used to test immunotherapies and elucidate treatment resistance mechanisms. The development of 3D in vitro melanoma models that faithfully replicate the complexity of the TME and its interaction with the immune system will provide us with the technical tools to better understand ICI resistance and increase its efficacy, thereby improving personalized melanoma therapy.

Cisplatin exhibits superiority over MMC as a perfusion agent in a peritoneal mesothelioma patient specific organoid HIPEC platform.

Peritoneal mesothelioma (PM) is a rare malignancy with poor prognosis, representing about 10-15% of all mesothelioma cases. Herein we apply PM patient-derived tumor organoids (PTOs) in elucidating personalized HIPEC responses to bypass rarity of disease in generating preclinical data. Specimens were obtained from PM patients undergoing cytoreductive surgery with HIPEC. PTOs were fabricated with tumor cells suspended in ECM-hydrogel and treated with HIPEC regimen parameters. Viability and characterization analyses were performed post-treatment. Treatment efficacy was defined as ≥ 50% viability reduction and p < 0.05 compared to controls. From October 2020 to November 2022, 17 tumors from 7 patients were biofabricated into organoids, with 16/17 (94.1%) sites undergoing comparative 37° and 42° treatments with cisplatin and mitomycin C (MMC). Hyperthermic cisplatin and MMC enhanced cytotoxicity which reduced treatment viability by 25% and 22%, respectively, compared to normothermia. Heated cisplatin displayed the greatest cytotoxicity, with efficacy in 12/16 (75%) tumors and an average viability of 38% (5-68%). Heated MMC demonstrated efficacy in 7/16 (43.8%) tumors with an average treatment viability of 51% (17-92.3%). PTOs fabricated from distinct anatomic sites exhibited site-specific variability in treatment responses. PM PTOs exhibit patient and anatomic location treatment responses suggestive of underlying disease clonality. In PM organoids cisplatin is superior to MMC in HIPEC.

Utilizing Patient-Derived Organoids in the Management of Colorectal Cancer with Peritoneal Metastases: A Review of Current Literature.

INTRODUCTION: Treatment of colorectal cancer-derived peritoneal carcinomatosis (CRC-PC) is challenging due to cellular heterogeneity that exhibits variable degrees of resistance to systemic as well as intraperitoneal chemotherapy. Therefore, it is not a surprise that the majority of patients undergoing cytoreductive surgery with HIPEC will experience recurrence. Patient-derived tumor organoids (PTOs) may be potentially capable of informing clinical treatment decisions at the level of the individual patient. In this study, we review the current landscape of CRC-PC PTO literature. METHODS: PubMed was queried for peer-reviewed publications studying CRC-PC organoids. Original articles which harnessed organoids as a research platform to study CRC-PC were included for review. Xenograft organoid studies were excluded. RESULTS: A total of 5 articles met inclusion criteria published between 2017 and 2022 and underwent complete analysis. Study topics included optimization of current therapies, identification of novel drug applications, and identification of disease mechanisms. Current therapies studied included systemic chemotherapy, targeted inhibitors, and HIPEC regimens. CONCLUSIONS: Patient-derived tumor organoids are a valuable personalized research tool that can complement real-time clinical settings. Additional research is needed to optimize methodologies of organoid incorporation in patients with colorectal cancer with peritoneal carcinomatosis.

Application of immune enhanced organoids in modeling personalized Merkel cell carcinoma research.

Merkel cell carcinoma (MCC) is a rare neuroendocrine cutaneous cancer, with incidence of less than 1/100,000, low survival rates and variable response to chemotherapy or immunotherapy. Herein we explore the application of patient tumor organoids (PTOs) in modeling personalized research in this rare malignancy. Unsorted and non-expanded MCC tumor cells were isolated from surgical specimens and suspended in an ECM based hydrogel, along with patient matched blood and lymph node tissue to generate immune enhanced organoids (iPTOs). Organoids were treated with chemotherapy or immunotherapy agents and efficacy was determined by post-treatment viability. Nine specimens from seven patients were recruited from December 2018-January 2022. Establishment rate was 88.8% (8/9) for PTOs and 77.8% (7/9) for iPTOs. Histology on matched patient tissues and PTOs demonstrated expression of MCC markers. Chemotherapy response was exhibited in 4/6 (66.6%) specimens with cisplatin and doxorubicin as the most effective agents (4/6 PTO sets) while immunotherapy was not effective in tested iPTO sets. Four specimens from two patients demonstrated resistance to pembrolizumab, correlating with the corresponding patient's treatment response. Routine establishment and immune enhancement of MCC PTOs is feasible directly from resected surgical specimens allowing for personalized research and exploration of treatment regimens in the preclinical setting.

Immuno-reactive cancer organoid model to assess effects of the microbiome on cancer immunotherapy.

Immune checkpoint blockade (ICB) therapy has demonstrated good efficacy in many cancer types. In cancers such as non-resectable advanced or metastatic triple-negative breast cancer (TNBC), it has recently been approved as a promising treatment. However, clinical data shows overall response rates (ORRs) from ~ 3-40% in breast cancer patients, depending on subtype, previous treatments, and mutation status. Composition of the host-microbiome has a significant role in cancer development and therapeutic responsiveness. Some bacterial families are conducive to oncogenesis and progression, while others aid innate and therapeutically induced anti-tumor immunity. Modeling microbiome effects on anti-tumor immunity in ex vivo systems is challenging, forcing the use of in vivo models, making it difficult to dissect direct effects on immune cells from combined effects on tumor and immune cells. We developed a novel immune-enhanced tumor organoid (iTO) system to study factors affecting ICB response. Using the 4T1 TNBC murine cell line and matched splenocytes, we demonstrated ICB-induced response. Further administration of bacterial-derived metabolites from species found in the immunomodulatory host-microbiome significantly increased ICB-induced apoptosis of tumor cells and altered immune cell receptor expression. These outcomes represent a method to isolate individual factors that alter ICB response and streamline the study of microbiome effects on ICB efficacy.

Pleural Effusion Aspirate for Use in 3D Lung Cancer Modeling and Chemotherapy Screening.

Lung cancer is the leading cause of cancer-related deaths worldwide, yet most currently used in vitro cancer models are confined to traditional 2D cell culture conditions. Recently; however, innovative 3D models such as tumor tissue equivalents (organoids) have been adopted by researchers to recapitulate tissue architecture and physiology in order to improve disease modeling and drug testing. We have hypothesized that 3D lung organoids, incorporating cells directly from patients, will enable personalized disease modeling and tumor cell characterization compared to traditional 2D cultures. Here, we discuss the fabrication of 3D lung cancer organoids using a rare cell source, pleural effusion aspirate. We tracked the phenotypic change that developed in short-term culturing and characterized the cell population within the organoids. We found that isolated patient cells embedded directly into organoids created anatomically relevant structures and displayed lung cancer specific behaviors compared to cultures that first grew in 2D conditions. Additionally, we compared responses of patient cells from pleural effusion aspirates to chemotherapy in 2D and 3D cell culture systems. Our results show that cells in 2D cultures are more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to isolate tumor cells from pleural effusion fluid of lung cancer patients and create organoids that display in vivo like anatomy and drug response. This technology can serve as a more accurate disease model for studying tumor progression and drug development.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

Exploiting maleimide-functionalized hyaluronan hydrogels to test cellular responses to physical and biochemical stimuli.

Currentin vitrothree-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine-glycine-aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factorß(TGF-ß) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell-matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ßto elicit a fibrosis-like state. In the presence of TGF-ßbiochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell-matrix interactions on cell phenotype and overall liver functionality.

Organoid Platform in Preclinical Investigation of Personalized Immunotherapy Efficacy in Appendiceal Cancer: Feasibility Study.

PURPOSE: Immunotherapy efficacy data on appendiceal cancer from clinical trials does not exist, due to appendiceal cancer incidence of 0.97 per 100,000. The goal of this study was to preclinically explore the application of immunotherapy in treating appendiceal cancer in a personalized organoid model. EXPERIMENTAL DESIGN: Patient tumor organoids (PTO) were fabricated using unsorted tumor cells with and without enrichment with patient-matched immune components derived from peripheral blood leukocytes, spleen, or lymph nodes [immune-enhanced PTOs (iPTO)]. Organoids were cultured for 7 days, followed by treatment with immunotherapy (pembrolizumab, ipilimumab, nivolumab), and assessed for treatment efficacy. RESULTS: Between September 2019 and May 2021, 26 patients were enrolled in the study. Successful testing was conducted in 19 of 26 (73.1%) patients, with 13 of 19 (68.4%) and 6 of 19 (31.6%) patients having low-grade appendiceal (LGA) and high-grade appendiceal (HGA) primaries, respectively. Immunotherapy response, with increased expression of granzyme B and cleaved caspase 3 and decreased expression of CK20 and ATP activity, was exhibited in 4 of 19 (21.1%) pembrolizumab-treated and 2 of 19 (10.5%) nivolumab-treated iPTOs. Post-immunotherapy cellular viability, in responding HGA organoids to pembrolizumab, decreased to less than 15% (P < 0.05). LGA iPTO treatment responses were observed in pembrolizumab and nivolumab, with an 8%-47.4% (P < 0.05) viability compared with controls. Ipilimumab showed no efficacy in the examined cohort. CONCLUSIONS: Immunotherapy shows measurable efficacy in appendiceal cancer organoids. Information derived from immunocompetent organoids may be applied in selecting patients for clinical trial enrollment in rare diseases where preclinical models of disease are lacking.

Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harshin vivoenvironments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

Cell Viability Assays in Three-Dimensional Hydrogels: A Comparative Study of Accuracy.

Three-dimensional (3D) cell culture systems, such as tumor organoids and multicellular tumor spheroids, have been developed in part as a result of major advances in tissue engineering and biofabrication techniques. 3D cell culture offers great capabilities in drug development, screening, testing, and precision medicine owing to its physiological accuracy. However, since the inception of 3D systems, few methods have been reported to successfully analyze cell viability quantitatively within hydrogel constructs. In this study, we describe and compare commercially available viability assays developed for two-dimensional (2D) applications for use in 3D constructs composed of organic, synthetic, or hybrid hydrogel formulations. We utilized Promega's CellTiter-Glo®, CellTiter-Glo 3D, and CellTiter 96® MTS Assay along with Thermo Fisher's PrestoBlue™ assay to determine if these assays can be used accurately in 3D systems. Compared with direct cell viability commonly used in 2D cell culture, our results show cellular health output inaccuracies among each assay in differing hydrogel formulations. Our results should inform researchers of potential errors when using cell viability measurements in 3D cultures and conclude that microscopic imaging should be used, in combination, for validation. Impact statement Three-dimensional (3D) tissue organoids models are a valuable tool not only for studying drug toxicity but also for understanding human embryonic development, intra-tissue morphogenesis, and mechanisms of disease. In cancer organoids, such 3D models may be used for preclinical chemotherapy screening and for understanding cell death and viability mechanisms under physiologically relevant conditions. Cell viability assays are necessary for assessing the effect of biological reagents on cellular health and have been used on in vitro cell cultures for many years. With the increase of 3D systems in cellular biology research to determine therapeutic efficacy, two-dimensional assays that measure cell viability are being used outside their intended use on 3D constructs. In this study, we assess the accuracy of using various commercially available cell viability assays on different 3D hydrogel constructs to help researchers understand expected variability in their experimentation along microscopic imaging validation.

High-throughput microscopy reveals the impact of multifactorial environmental perturbations on colorectal cancer cell growth.

BACKGROUND: Colorectal cancer (CRC) mortality is principally due to metastatic disease, with the most frequent organ of metastasis being the liver. Biochemical and mechanical factors residing in the tumor microenvironment are considered to play a pivotal role in metastatic growth and response to therapy. However, it is difficult to study the tumor microenvironment systematically owing to a lack of fully controlled model systems that can be investigated in rigorous detail. RESULTS: We present a quantitative imaging dataset of CRC cell growth dynamics influenced by in vivo-mimicking conditions. They consist of tumor cells grown in various biochemical and biomechanical microenvironmental contexts. These contexts include varying oxygen and drug concentrations, and growth on conventional stiff plastic, softer matrices, and bioengineered acellular liver extracellular matrix. Growth rate analyses under these conditions were performed via the cell phenotype digitizer (CellPD). CONCLUSIONS: Our data indicate that the growth of highly aggressive HCT116 cells is affected by oxygen, substrate stiffness, and liver extracellular matrix. In addition, hypoxia has a protective effect against oxaliplatin-induced cytotoxicity on plastic and liver extracellular matrix. This expansive dataset of CRC cell growth measurements under in situ relevant environmental perturbations provides insights into critical tumor microenvironment features contributing to metastatic seeding and tumor growth. Such insights are essential to dynamical modeling and understanding the multicellular tumor-stroma dynamics that contribute to metastatic colonization. It also establishes a benchmark dataset for training and testing data-driven dynamical models of cancer cell lines and therapeutic response in a variety of microenvironmental conditions.

Semiconducting polymer nanoparticles for photothermal ablation of colorectal cancer organoids.

Colorectal cancer (CRC) treatment is currently hindered by micrometastatic relapse that cannot be removed completely during surgery and is often chemotherapy resistant. Targeted theranostic nanoparticles (NPs) that can produce heat for ablation and enable tumor visualization via their fluorescence offer advantages for detection and treatment of disseminated small nodules. A major hurdle in clinical translation of nanoparticles is their interaction with the 3D tumor microenvironment. To address this problem tumor organoid technology was used to evaluate the ablative potential of CD44-targeted polymer nanoparticles using hyaluronic acid (HA) as the targeting agent and coating it onto hybrid donor acceptor polymer particles (HDAPPs) to form HA-HDAPPs. Additionally, nanoparticles composed from only the photothermal polymer, poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe), were also coated with HA, to form HA-BSe NPs, and evaluated in 3D. Monitoring of nanoparticle transport in 3D organoids revealed uniform diffusion of non-targeted HDAPPs in comparison to attenuated diffusion of HA-HDAPPs due to nanoparticle-matrix interactions. Computational diffusion profiles suggested that HA-HDAPPs transport may not be accounted for by diffusion alone, which is indicative of nanoparticle/cell matrix interactions. Photothermal activation revealed that only HA-BSe NPs were able to significantly reduce tumor cell viability in the organoids. Despite limited transport of the CD44-targeted theranostic nanoparticles, their targeted retention provides increased heat for enhanced photothermal ablation in 3D, which is beneficial for assessing nanoparticle therapies prior to in vivo testing.

Manipulating the Tumor Microenvironment in Tumor Organoids Induces Phenotypic Changes and Chemoresistance.

Tumors comprised a tightly surrounded tumor microenvironment, made up of non-cellular extracellular matrix (ECM) and stromal cells. Although treatment response is often attributed to tumor heterogeneity, progression and malignancy are profoundly influenced by tumor cell interactions with the surrounding ECM. Here, we used a tumor organoid model, consisting of hepatic stellate cells (HSCs) embedded in collagen type 1 (Col1) and colorectal cancer cell (HCT-116) spheroids, to determine the relationship between the ECM architecture, cancer cell malignancy, and chemoresistance. Exogenous transforming growth factor beta (TGF-ß) used to activate the HSCs increased the remodeling and bundling of Col1 in the ECM around the cancer spheroid. A dense ECM architecture inhibited tumor cell growth, reversed their mesenchymal phenotype, preserved stem cell population, and reduced chemotherapy response. Overall, our results demonstrate that controlled biofabrication and manipulation of the ECM in tumor organoids results enables studying tumor cell-ECM interactions and better understand tumor cell response to chemotherapies.

Personalized Identification of Optimal HIPEC Perfusion Protocol in Patient-Derived Tumor Organoid Platform.

BACKGROUND: Chemotherapy dosing duration and perfusion temperature vary significantly in HIPEC protocols. This study investigates patient-derived tumor organoids as a platform to identify the most efficacious perfusion protocol in a personalized approach. PATIENTS AND METHODS: Peritoneal tumor tissue from 15 appendiceal and 8 colon cancer patients who underwent CRS/HIPEC were used for personalized organoid development. Organoids were perfused in parallel at 37 and 42 °C with low- and high-dose oxaliplatin (200 mg/m2 over 2 h vs. 460 mg/m2 over 30 min) and MMC (40 mg/3L over 2 h). Viability assays were performed and pooled for statistical analysis. RESULTS: An adequate organoid number was generated for 75% (6/8) of colon and 73% (11/15) of appendiceal patients. All 42 °C treatments displayed lower viability than 37 °C treatments. On pooled analysis, MMC and 200 mg/m2 oxaliplatin displayed no treatment difference for either appendiceal or colon organoids (19% vs. 25%, p = 0.22 and 27% vs. 31%, p = 0.55, respectively), whereas heated MMC was superior to 460 mg/m2 oxaliplatin in both primaries (19% vs. 54%, p < 0.001 and 27% vs. 53%, p = 0.002, respectively). In both appendiceal and colon tumor organoids, heated 200 mg/m2 oxaliplatin displayed increased cytotoxicity as compared with 460 mg/m2 oxaliplatin (25% vs. 54%, p < 0.001 and 31% vs. 53%, p = 0.008, respectively). CONCLUSIONS: Organoids treated with MMC or 200 mg/m2 heated oxaliplatin for 2 h displayed increased susceptibility in comparison with 30-min 460 mg/m2 oxaliplatin. Optimal perfusion protocol varies among patients, and organoid technology may offer a platform for tailoring HIPEC conditions to the individual patient level.

Simulating the human colorectal cancer microenvironment in 3D tumor-stroma co-cultures in vitro and in vivo.

The tumor microenvironment (TME) plays a significant role in cancer progression and thus modeling it will advance our understanding of cancer growth dynamics and response to therapies. Most in vitro models are not exposed to intact body physiology, and at the same time, fail to recapitulate the extensive features of the tumor stroma. Conversely, animal models do not accurately capture the human tumor architecture. We address these deficiencies with biofabricated colorectal cancer (CRC) tissue equivalents, which are built to replicate architectural features of biopsied CRC tissue. Our data shows that tumor-stroma co-cultures consisting of aligned extracellular matrix (ECM) fibers and ordered micro-architecture induced an epithelial phenotype in CRC cells while disordered ECM drove a mesenchymal phenotype, similar to well and poorly differentiated tumors, respectively. Importantly, co-cultures studied in vitro, and upon implantation in mice, revealed similar tumor growth dynamics and retention of architectural features for 28 days. Altogether, these results are the first demonstration of replicating human tumor ECM architecture in ex vivo and in vivo cultures.